The Clinical And Basic Research Of Hepatic Hereditary Hemorrhagic Telangiectasia

Background and SignificanceHereditary hemorrhagic telangiectasia (HHT), also known as Osler-Weber-Rendu syndrome, which is characterized by the skin mucosa telangiectasias of the whole body (such as nose, oral cavity, lip, tongue, fingers) and visceral arteriovenous malformations (liver, pulmonary, brain, gastrointestinal tract and so on), is a systemic angiogenesis disorder caused by the mutation of genes related to transforming growth factor beta signal pathway. HHT is an autosomal dominant disease, and to date, there is no report of a genetically confirmed case of homozygosity in human HHT. The heterozygous patients of HHT could present a wide range of individual symptoms, which could develop progressively with increasing age, and the underlying mechanism is "haploinsufficiency ". The involved organ or tissue, the clinical features, and the outcome or prognosis of HHT patients vary greatly among the affected people, so that it is difficult for early diagnosis and treatment. In2002, a genetic epidemiologic study of HHT in the Akita prefecture (population1.2million) located in northern Japan was reported, and the estimated population incidence of this genetic vascular disorder in the Japanese county was1:8000-1:5000, roughly comparable with those reported in European and American populations, which is contradictory to the traditional view that HHT is rare among Asians.A low prevalence of liver involvement, ranging between8%and31%, has been reported in retrospective studies performed during the1980s and1990s in patient series selected according to clinical criteria. However, liver vascular malformations of HHT are being discovered fortuitously more frequently with the increasing performance of sensitive imaging techniques such as color Doppler ultrasound (US) or multislice computed tomography (CT) and the improvement method of screening. The incidence of liver involvement has been shown to be higher (41.4%) in a family screening protocol study and to range between67%and84%in unselected consecutive HHT patients. The difference in the rate of liver involvement in consecutive patient series of HHT may be influenced by the criteria used for the diagnosis. It is obviously that the study of the liver involvement of HHT becomes one of the most important parts of the HHT research and the concept of "Hepatic Hereditary Hemorrhagic Telangiectasia (HHHT)" is accepted gradually.The majority HHHT patients are asymptomatic onset, without any special signs, and the proportion of patients present with typical symptoms (such as high-output heart failure, portal hypertension and biliary disease) who seek help from doctors is very low. The clinical diagnosis of HHT has been based upon the Curacao criteria, however, it should be noted that the criteria represent consensus expert opinion, and its sensitivity and specificity have not been formally validated. Gene detection has been applied to screen for the HHT diagnosis, but more and more mutation sites were found, and new virulence genes were reported continually. Due to the family specific genetic characteristics of HHT, the gene diagnosis is difficult for the patients with unknown mutant gene. In a word, the diagnostic strategy for HHHT needs to be perfected. With respect to the treatment of HHHT, the appropriate therapy, especially the surgical therapy is controversial. The surgical treatment of HHHT consists of hepatic artery embolization, open ligation or banding related to the feeding artery of the hepatic arteriovenous malformation, and liver transplantation. Embolization, ligation and banding have been performed in patients with controversial results and with the development of complications derived from ischemia of the biliary tree. What is more, these methods may cause hepatic or biliary necrosis. One article, published in Annals of Surgery in2006, summarize the effect of liver transplantation from14centers where40patients were transplanted for HHT during the period from1985to2003. Among them, Twenty-four (60%) patients had post-transplant complications, eight (20%) patients died perioperatively, six of them due to bleeding, one due to cardiac failure, and the last one died late due to chronic rejection. In addition, two patients were diagnosed with possible recurrences.In recent years, the case reports about HHHT in China arise gradually, unfortunately, there is no systemic cases analysis and clinical study. We visited and made follow-up of the HHHT patients in Shandong province (such as Huantai county, Shenxian county, Mengyin county), Zhejiang province (Quzhou city), Henan province (Zhengzhou city), Hebei province (Shijiazhuang city) and Shanghai to collect clinical data and pedigrees. In the first part of this doctoral dissertation, the clinical data of15HHHT patients from Qilu Hospital of Shandong University and cooperation units from Shandong province, Henan province, Hebei province and Zhejiang province were retrospectively analyzed. We summarized the clinical manifestation, imaging and laboratory features, and investigated the diagnostic strategy and individual treatment.Hereditary hemorrhagic telangiectasia (HHT) is a disease of inherited vascular dysplasia, and its genetic background is the mutations of the coding genes of transforming growth factor beta signal pathway. The two identified virulence genes are ENG/endoglin (HHT type1) on chromosome9q33-34, and activin receptor-like kinase1(ALK-1)(HHT type2) on chromosome12q13. ENG codes for a homodimeric integral membrane glycoprotein, which takes part in the form of the receptor complex of TGF-β. ALK-1gene codes for the activin-like kinase receptor type Ⅰ of the TGF-β receptor superfamily. Both ENG and ALK-1express mainly restricted to endothelial and vascular smooth muscle cells, and play very important role in angiogenesis. To date, four other genes are regarded as the suspected virulence genes, two of which with unknown function. The other two(MADH4,BMPR2) are involved in the transforming growth factor beta signal pathway too.TGF-β superfamily plays a pivotal role in cellular proliferation, differentiation, embryo development and vascular formation. TGF-β family members initiate their activities on the cell by binding to a heteromeric complex of type Ⅰ and type Ⅱ serine/threonine kinase receptors at the cell surface. Binding of the ligand to the type Ⅱ receptor recruits the type I receptor that becomes phosphorylated and itself phosphorylates members of the Smad protein family. Once Smad proteins have been activated they are translocated into the nucleus and regulate gene expression as transcription factors. The mutation of ENG, ALK-1and MADH4lead to the abnormality of the receptor and Smad4of TGF-β signal pathway, which causes the disorder of angiogenesis.According to the mechanism of HHT, we assume that the method of up-regulating TGF-β signalling, especially the mode of enhancing the function of receptor or Smad4, may slow down the progression of HHT. RNF11may be the key of positive regulation factor of transforming growth factor beta signal pathway.The gene of Ring-Finger protein11(RNF11) is originally cloned from a library enriched for tumour cDNAs. It is reported that nearly80protein interactions for RNF11and RNF11is placed at the cross-roads of cell signalling and transcriptional regulation. It modulats TGF-β signal through the direct action of itself and the indirect action of ubiquitination. RNF11is known to interact with both E2conjugating enzymes and E3ubiquitination ligases (such as Smurf1,Smurf2). Smurf2is a highly effective negative regulator of the TGF-β pathway:on the one hand, Smurf2can interact with Smad2, thereby targeting Smad2for degradation, as well as the Smad2associated transcription factor SnoN; on the other hand, Smurf2has been shown to interact with Smad7and target the TGF-β receptor for degradation. Foreign scholars infer that RNF11can up-regulate the TGF-β through abrogating Smurf2-mediated ubiquitination of the TGF-β receptor and enhancing Smad2-Smad4transcriptional activity.We chose Ring-Finger protein11(RNF11) as the regulator of TGF-β signal pathway in order to slow down the progression of HHT. In the second part of this doctoral dissertation, we constructed the eukaryotic expression vector of RNF11, and then transfected the vector into the Chang Liver cell, which was a cell line of non-tumor hepatocyte, in order to observe whether RNF11could enhance the effect of TGF-β and suppress the proliferation of this cell line. The purpose of this work is to test and verify the positive regulation effect of RNF11towards the TGF-β signal pathway and to prepare for the experimental research for application of TGF-β signaling regulator in order to regulate the angiogenesis and treat HHHT. Part One The Diagnostic Strategy and Individual Treatment Of Hepatic Hereditary Hemorrhagic TelangiectasiaObjectiveTo analyze the clinical features of the Hepatic Hereditary Hemorrhagic Telangiectasia(HHHT) in order to improve the current understanding of the disease and explore the diagnostic strategy and treatment.MethodThe clinical data of15HHHT patients were retrospectively analyzed. The clinical manifestations, features of imaging and laboratory examination were summarized, and the diagnosis, treatment and prognosis of the disease were investigated.Results1. HHHT had nonspecific symptoms, some patients presented with right upper quadrant discomfort, shortness of breath, liver bruit or anemia. The condition of HHHT patients could be worsened by liver cirrhosis or portal hypertension rapidly.2. The results of ultrasonography and CT showed parenchymal hepatic telangiectasias, intrahepatic shunting and at least one enlarged hepatic artery in all of the15patients. Digtial subtraction angiography (DSA) was not clear enough for the two complex HHHT patients with more than one enlarged hepatic arteries, but computed tomographic angiography (CTA) was feasible.3. The specimens showed diffuse intraparenchymal telangiectasias, ectatic vessels, and alternating areas of regeneration and atrophy. Some areas of the specimen were cirrhotic, and showed nodular hyperplasia with fibrous tissue along ectatic vessels. Immunohistochemistry experiment showed the liver tissue of HHHT patients presented with strong positive feature of TGF-β1.4. According to the degree and stages of the disease, the patients could be divided into asymptomatic HHHT, simple HHHT and complex HHHT. Among the six patients who underwent surgical treatment, five patients accepted ligation/banding of the enlarged hepatic arteries with subsequent disappearance of symptoms. Three cases underwent interventional treatment, but one complex patient with multiple hepatic arteries failed. The patient did not accept surgical treatment and died30months later. The ultrasonography and/or CT of four patients with medical therapy showed the pathological changes were aggravated gradually. Two HHHT patients with one enlarged hepatic artery, who were asymptomatic at first, suffered liver dysfunction and ascites21and35months later respectively, and one of them died6months later.ConclusionTelangiectasias, intrahepatic arteriovenous and hepatic aneurysm are the main imaging characteristics of HHHT, and imaging diagnosis has significant value in the diagnosis of HHHT. HHHT is a progressive disease, and individualized surgical procedures are proper, while early and active therapy may be helpful. Open ligation/banding are effective for HHHT patients.SignificanceThe clinical data of patients in China were summarized and analyzed systematically, and we brought up the idea of diagnostic strategy and individual treatment firstly. With respect to the postoperative complications of ischemia of hepatic tissue and biliary tree after ligation, we proposed the preventive measures or the modified operation. Part Two Experimental Study for Ring-Finger Protein11(RNF11) and Transforming Growth Factor β Conductive PathwayObjectiveTo explore the effect of the Ring-Finger Protein11(RNF11) towards the proliferation of Chang Liver through the transforming growth factor beta (TGF-β) signal pathway, and to test and verify the positive regulation effect of RNF11towards the TGF-β signalling.MethodsThe expression levels of RNF11in Chang Liver cell, Hepg-2cell, A549cell were determined by RT-PCR. The Chang Liver cell was transfected with eukaryotic expression vector pCMV6-RNF11-GFP in mediation of liposomes.5ng/ml TGF-P1in final concentration was administrated into the Chang Liver cell of the group A of transfection cells, the group B of empty plasmid cell and the group C of non-transfection cell24hours later. We observed the cell morphology and growth condition with microscope. Cell regeneration was detected through the method of Brdu-DAPI labeling, Cell Counting Kit-8measure and cell growth curve description at different time spots after the administration of TGF-β1.Results1. Chang Liver cell expressed the RNF11.2. The morphology of transfected Chang Liver cell (group A) was changed, and cell condition was not good. The shape of cells both in group B of empty plasmid cell and group C of non-transfection cell was normal. The Cell Counting Kit-8measure and cell growth curve description showed the proliferation ability of the transfected cells was suppressed compared with the group B of empty plasmid cell and the group C of non-transfection.3. The cell inhibition rate of group A (transfection group)24hours,48hours and72hours after treatment of TGF-01were10.80%.14.47%.16.31%respectively, and the result of the group B (empty-transfection group) were3.52%,4.61%,4.84%respectively.4. Brdu-DAPI labeling method indicated the proportion of the Brdu-positive cell was decreased with statistical significance (P<0.05).ConclusionsRNF11takes role in the process of hepatocytes proliferation in vitro, and it could suppress proliferation through TGF-βsignalling. RNF11is a positive regulator of TGF-β signal pathway.SignificanceRNF11was placed at the cross-roads of cell signalling and transcriptional regulation, and we tested and verified the positive regulation effect of RNF11towards the TGF-β signalling. What's more, the pathogeny of HHHT was the mutation of related genes of TGF-β signal pathway. According to the mechanism of "haploinsufficiency", we assumed that the method of up-regulating TGF β signalling, especially the mode of enhancing the function of receptor or Smad4, may slow down the progression of HHT.